This invention relates to the cell culture production of the thrombolytic agent tissue plasminogen activator. More particularly, the invention relates to a method for increasing the yield of tissue plasminogen activator in culture of mammalian cells.
It is known that various plasminogen activators (PA) are widely distributed throughout the body and can be purified from tissue extracts. Typical examples of tissue sources are kidney, lung and uterus tissues. The best characterized of these plasminogen activators fall into two major groups, urokinase plasminogen activator (u-PA) and tissue plasminogen activator (t-PA). u-PA and t-PA are present in ng/ml concentrations in human plasma and are immunologically unrelated. t-PA has been demonstrated to have higher affinity for fibrin than u-PA. u-PA products isolated and purified from human urine and from mammalian kidney cells are pharmaceutically available as thrombolytic agents.
Due to the extremely low concentration of t-PA in blood and tissue extracts, other sources and means of producing this preferred thrombolytic agent have been sought after.
One method of producing t-PA on a large scale comprises isolating the protein from the culture fluid of human melanoma cells grown under in vitro cell culture conditions. An established human melanoma cell line (Bowes) has been used for this purpose. See, for example, European Patent Application No. 41,766, published Dec. 16, 1981; Rijken and Collen, J. Biol. Chem. 256(13), 7035-7041 (1981); and Kluft et al., Adv. Biotech. Proc. 2, Alan R. Liss, Inc., 1983, pp. 97-110. The Bowes melanoma t-PA is a glycoprotein which has a molecular weight of about 68,000-70,000 daltons and a 527 amino acid structure with serine at the NH.sub.2 -terminus. The melanoma t-PA can exist as two chains, an A-chain and a B-chain. It also separates into two variants (or isoforms) in the A-chain, known as types I and II which differ by about M.sub.r 2000-3000. See Ranby et al., FEBS Lett. 146 (2), 289-292 (1982), and Wallen et al., Eur. J. Biochem. 132, 681-686 (1983). Type I is glycosylated at Asn-117, Asn-184 and Asn-448 whereas Type II is glycosylated only at Asn-117 and Asn-448 according to Pohl et al., Biochemistry 23, 3701-3707 (1984). A high mannose structure has been assigned to Asn-117 whereas two complex carbohydrate structures are assigned to Asn-184 and Asn-448 by Pohl et al., "EMBO Workshop on Plasminogen Activators," Amalfi, Italy, Oct. 14-18, 1985.
Genetic information from the Bowes melanoma cell line also has been embodied in E. coli by conventional recombinant DNA gene splicing methods to permit the production of the t-PA protein moiety by that microorganism. See, for example, UK Patent Application No. 2,119,804, published Nov. 23, 1983; Pennica et al., Nature 301, 214-221 (1983); and Vehar et al., Bio/Technology 2 (12), 1051-1057 (1984). Recombinant t-PA produced by the expression of Bowes melanoma genetic material in cultured mammalian cells has been administered to humans with some measure of effectiveness. See Collen et al., Circulation 70(16), 1012-1017 (1984).
The recombinant-derived t-PA produced in E. coli is non-glycosylated and contains only the protein moiety of t-PA. Although the specific function of the carbohydrate moiety on t-PA has not been determined, it is known, in general, that glycosylation can cause certain differences of which the following are of biological interest: antigenicity, stability, solubility and tertiary structure. The carbohydrate side-chains also can affect the protein's half-life and target it to receptors on the appropriate cells. See, for example, Delente, Trends in Biotech. 3(9), 218 (1985), and Van Brunt, Bio/Technology 4, 835-839 (1986). The functional properties of carbohydrate-depleted t-PA are further discussed by Little, et al., Biochemistry 23, 6191-6195 (1984), and by Opdenakker et al., "EMBO workshop on Plasminogen Activators," Amalfi, Italy, Oct. 14-18, 1985. The latter scientists report that enzymatic cleavage of carbohydrate side-chains from the melanoma (Bowes) derived t-PA by treatment with .alpha.-mannosidase causes an increase in the biologic activity of the modified t-PA.
Cultured normal human cells also have been used as a source of t-PA as can be seen from U.S. Pat. Nos. 4,335,215, 4,505,893, 4,537,860, and 4,550,080. Various cell sources mentioned in said patents are primary embryonic (or fetal) kidney, lung, foreskin, skin and small intestines (Flow Laboratories) or the AG1523 cell line. Brouty-Boye et al., Bio/Technology 2 (12), 1058-1062 (1984), further disclose the use of normal human embryonic lung cells for the production of t-PA. Rijken and Collen, J. Biol. Chem. 256(13), 7035-7041 (1981), and Pohl et al., FEBS Lett. 168(1), 29-32 (1984), disclose the use of human uterine tissue as a t-PA source material. European Patent Application No. 236,289, published Sept. 9, 1987, describes a uniquely glycosylated t-PA derived from normal human colon fibroblast cells.
Production of glycosylated t-PA in non-human mammalian cells also is known. Thus, Kaufman et al., Mol. Cell. Biol. 5, 1750-1759 (1985), and European Patent Application No. 117,059, published Aug. 29, 1984, describe the use of Chinese hamster ovary cells and Browne et al., Gene 33, 279-284 (1985), describe the use of mouse L cells for such production. Kaufman et al., state that the Chinese hamster ovary t-PA is glycosylated in a similar but not identical manner as native t-PA. Glycosylated forms of t-PA obtained by recombinant DNA are further described by Zamarron et al., J. Biol. Chem. 259 (4), 2080-2083 (1984), and Collen et al., J. Pharmacol. Expertl. Therap. 231 (1), 146-152 (1984).
Notwithstanding the advantages in the production of t-PA by culture of mammalian cells, it has been found that such production is regulated by negative feedback which in turn is controlled by the concentration of the extracellular t-PA. Various methods have been reported heretofore to minimize the effects of the negative feedback on the biosynthesis of t-PA. One method is to use a high ratio of medium volume to cell number so that the extracellular t-PA does not reach a high concentration. Another method is to perfuse the system continuously, thereby removing part of the t-PA containing medium and replacing it with fresh medium. Still another method is to adsorb the product continuously and to recycle the supernatant. See Kadouri and Bohak in Adv. Biotechnological Proc. 5, Eds. Mizrahi and van Wesel, Alan R. Liss, Inc., 1985, pp. 275-299.